The present invention relates to method and apparatus for measuring concentration of a subject, particularly applicable to concentration measurement for microorganism or product thereof in food chemistry.
During the process of cultivating various microorganisms conventionally utilized for mass cultivation of microorganisms, collection of their products and the other purposes, it is well known to determine concentrations of such microorganisms or their products and thereby to achieve proper control of their alimentation and allowable concentrations in respective culture fluids.
Conventionally, the microorganism concentration in a culture fluid has been determined according to a basic procedure in which a portion of the culture fluid is sampled and this sample is subjected to various methods of measuring the concentration, for example, the method based on dry weight of microorganism, the nephelomatic method and the method based on microorganism population counts.
However, such basic procedure necessarily requires much time and labor, on one hand, and is practically unsuitable for the in-line measurement from the viewpoint of undesirable contamination, on the other hand.
Accordingly, there has been a serious demand for method and apparatus allowing a germless in-line measurement to be continuously performed on real time for improving efficiency of cultivation.
As the methods prior art allowing the instrumental measurement of in-line manner, (A) optical method of measurement, (B) reactive method of measurement and (C) electrochemical method of measurement have already been disclosed.
(A) Optical method of measurement using an optical apparatus comprising a sensor containing therein a light emitter and a light receptor:
Japanese Utility Model Laid-Open Application No. 1987-16457 disclose an apparatus including a light receptor adapted to convert changes in quantity of received light to changes in quantity of electricity which are, in turn, operationally processed to determine concentrations of a subject. This apparatus is of a compact structure, no interfering with circulation of the subject fluid and useful even in environment of high temperature and pressure.
Japanese Patent Laid-Open Application No. 1976-49787 discloses an apparatus for measuring concentration of microorganism or the like in a culture fluid by determining quantity of transmitted light via optical fibre. With this apparatus any microorganism sticking to the apparatus wall may be killed utilizing UV rays and said optical fibre is housed in a container provided with an openable cover so as to protect the optical fibre against adverse effect of external light and bubbles.
(B) Reactive method of measurement:
Japanese Patent Laid-Open Application No. 1987-64934 discloses a biosensor in the form of quartz oscillator having antibody immobilized on its electrode surfaces for detection and concentration measurement of microorganisms.
Japanese Patent Laid-Open Application No. 1975-36198 discloses a temperature-sensitive apparatus including a probe coated with a microorganism or enzyme to determine concentration of a molecule as the substrate of this microorganism or enzyme.
(C) Electrochemical method of measurement:
Japanese Patent Laid-Open Application No. 1985-135754 discloses an apparatus for measuring concentration of a subject. The apparatus includes a working electrode and an opposite electrode both located in a passage of solvent and portion of this passage that surround said electrodes, respectively, are of variable diameters. Concentration of a subject is determined on the basis of changes in quantity of electricity generated between said electrodes.
Japanese Patent Laid-Open Application No. 1984-81551 discloses an arrangement comprising a pair of electrodes immersed in suspension of cells and periodical potential is applied to said electrodes so as to generate an electric current. The number of cells can be determined from a value of said electric current, in view of a fact that an electric current is generated as a living cell comes in direct contact with the electrode.
Japanese Patent Laid-Open Application No. 1986-48755 discloses a system adapted to measure an electric conductivity of given culture fluid and thereby to maintain a concentration of this culture fluid a the optimum level, utilizing a phase detector for the unbalanced output from an AC bridge including a pair of electrodes placed in the culture fluid of a reference concentration and a pair of electrodes placed in a passage of the culture fluid.
However, these arrangements of prior art as have been set forth above have unsolved problems as will be described. Although the in-line measurement is essential for the closed culture system typically such as the optical apparatus for measurement as above-mentioned in (A), it has been impossible for the prior art to prevent microorganism from sticking to the sensor. In consequence, the adverse influence upon the quantity of light becomes more significant and the measuring ability is correspondingly lowered as the amount of microorganism sticking to the light emitting and receiving surfaces increases. Additionally, particularly when the medium is initially colored or progressively colored as a cultivating time elapses, transmission of light must be adversely affected. Furthermore, external light rays such as those of illumination entering through a liquid level monitoring window also adversely affect the quantity of light emitting from the light emitter. These factors may often cause false measurements.
When the medium has a relatively high viscosity, such medium also tends to stick to the light emitting and receiving surfaces due to unique configurations of the light emitter and the light receptor, making it difficult to determine the state of the medium varying thus moment by moment. Moreover, portions of the medium and the microorganism that have higher concentrations with respect to the remainders cause increased measurement errors and therefore cannot be used for the measuring purpose.
For the reactive measuring apparatus as above-mentioned in (B), the cultivation by high temperature/high pressure processing is impossible and vibration of the apparatus must be avoided because these factors might destroy the carriers immobilized on the surface of the sensor or separate microorganism, enzyme or the like from the probe. A portion of the microorganism coupled to the antibody immobilized on the sensor is not available for production and reuse of the sensor requires not only washing operation but also the other various retreatment of the used sensor, for example, immobilization of the antibody thereon, to restore its original ability.
Said reactive measuring apparatus has its application limited to enzyme or microorganism of the type that requires reaction of a reactant or catalytic action provided by such reaction and said enzyme or microorganism, if it is of a high concentration, will restrict said catalytic action. Thus, a measurable range is correspondingly limited.
Finally, the electrochemical apparatus for measurement as above-mentioned in (C) has several disadvantages. Enzyme, microorganism or the like sticking to the electrodes may cause electric troubles which may cause, in turn, a false measurement. When it is desired to wash the electrodes, types of washing that can be safely used are limited from the viewpoint of undesirable results such as corrosion. In addition, the electrodes must be detached from the apparatus before they are washed, in order to wash them reliably and satisfactorily. While proper washing is essential for the electrochemical apparatus for measurement in order to maintain the apparatus in a germless condition, such proper washing requires troublesome operation and, therefore, the electrochemical apparatus is unsuitable for a system adapted for cultivation for a long period.
The respective prior arts (A), (B), (C) have the above-mentioned problems to be solved and one of the problems common to these prior arts is the problem of bubbles inevitably generated from the medium. The bubbles, if they stick to the sensor, will lower the detecting ability of the sensor and thereby make a reliable measurement difficult. Particularly in the apparatus including the electrodes, these bubbles sticking to the electrodes may often cause an electrolytic corrosion.